1. Field of the Invention
This invention relates generally to the field of immobilized proteins, and specifically with immunological binding partners which have been immobilized onto support materials. These support materials are primarily inorganic and have the property of contact activation of blood proteins.
2. Description of the Prior Art
The development of procedures to immobilize antibodies, other proteins and haptens onto a solid phase has provided extremely useful analytical methods for detecting and quantitating various organic substances of medical, environmental and industrial importance which appear in liquid mediums at very low concentrations. Diagnostic test kits which embody these procedures allow rapid and convenient tests to be performed. During use of a test kit a biological fluid that contains analyte at unknown concentration is usually combined with a solid phase support and with other chemical substances. The solid phase surfaces used in these diagnostic test kits frequently contain immobilized antigen (hapten) or, antibody that participates in competitive binding reactions or binding pair formation reactions. These binding reactions precede a signal development reaction in which the concentration of analyte to be detected is correlated to a change in a measurable quantity such as a light signal or electrical current.
Substances that participate in binding pair formation reactions are termed binding partners. Examples of such binding partners are avidin, biotin, antibody fragments, and haptens. The binding reactions are characterized by high affinity (strong association) between binding partners. The association constants for these reactions may be as high as 10.sup.10. The association between binding partners has to be controlled so that desorption of binding partner from the solid support is minimized. One aim of the art is to minimize this desorption.
Proteins and other types of binding partners have been immobilized or fixed on a wide variety of materials, both organic and inorganic. For example, antibodies and other proteins have been covalently immobilized to organic polymers by diazotization, amide bond formation, Schiff's base formation, Ugi reaction, amidination reactions and by crosslinking the protein in place with agents such as glutaraldehyde. Non-covalent techniques have also been used to couple substances to the surfaces of organic materials. For example, plastic surfaces may be coated with defined compositions of hydrophobic amino acids and then further treated with covalent coupling compounds as taught by Gadow and Wood in U.S. Pat. No. 4,657,873 issued (4/14/87) and entitled "Preactivated Plastics Surfaces for Immobilizing Organo-chemical and Biologic Materials". A brief summary of protein immobilization techniques may be found in Solid Phase Biochemistry, (1983) pp. 253-291 (ed. William H. Scouten).
Although most solid phase materials used in immunodiagnostics utilize antibody, antibody fragment, or hapten coupled to supports made of organic polymers, inorganic supports are sometimes preferable. Glass is especially desirable as a support medium because of its optical and mechanical properties and because it is chemically inert. Glass tubes in particular are desirable because of their relatively low cost and adaptability as cuvettes directly to colorimeters and nephelometers commonly available on the market. Glass fiber is used in some diagnostic procedures as a solid phase filter to immobilize red blood cells and other particles because of its mechanical rigidity and chemical inertness.
Unfortunately, the desirable chemical inertness of inorganic materials makes it difficult to immobilize molecular binding partners to them. Procedures used to covalently attach binding partners to inorganic materials require chemical modification of the surface to generate active residues that can in turn be chemically coupled to protein or other biologically active substances. For example, see U.S. Pat. No. 3,519,538 to Messing et al. issued (7/7/70) and entitled "Chemically Coupled Enzymes" disclosing enzymes bound via silanes and U.S. Pat. No. 3,652,761 to Weetall issued (3/28/72) and entitled "Immunochemical Composites and Antigen or Antibody Purification Therewith" disclosing antibodies bound via silanes. Messing and Odstrchel made a significant improvement to this early prior art by reacting siliceous support material with /-dianisidine and washing prior to contacting the activated support with protein solution (U.S. Pat. No. 3,983,000) issued (9/28/76) and entitled "Bonding Proteins to Inorganic Supports". Although an improvement, this procedure still requires multiple incubation and wash steps prior to contact of the inorganic support with a desired molecular binding partner. Coating of support material with a solution as taught by Schall, Jr. in U.S. Pat. No. 4,363,634 issued (10/18/83) and entitled "Glass Support Coated with Synthetic Polymer for Bioprocess" is applicable to glass supports but this procedure requires a separate coating step with polymeric film to form a surface layer on the glass and also a separate curing step of that film.
It cannot be overemphasized that the complexity of coating the solid support with molecular binding partners is a major determinant to the cost and manufacturing reproducibility (quality) of the diagnostic system. Ideally, one would like to simply contact a support with a solution that contains the binding partner to be adsorbed and then dry it. Passive adsorption of protein to glass prepared by acid washing was taught by Messing in U.S. Pat. No. 3,556,945 issued (1/19/71) and entitled "Enzyme Stabilization". In this passive adsorption procedure the glass surface is pretreated with acid, the passive adsorption step requires from 1 to 72 hours of incubation and the adsorption step is followed by a lengthy leaching step that may be a matter of hours and even up to many days. Furthermore, the desorption of protein from glass surfaces is a common and well recognized problem when prepared glass surfaces come into contact with blood proteins. For example, Vroman et al. reported the desorption of fibrinogin that had passively adsorbed onto "glass like" surfaces in Blood. 55, 156-159 (1980) and Brash and Samak discovered adsorbed serum albumin to become confromationally altered and slowly diffuse into solution from solid surfaces (J. Colloid Interface Sci. 65, 495-504 (1978). In some cases it was shown that although the total amount of protein adsorbed onto surfaces remains the same, there is exchange with soluble protein in solution which contacts the surface. For an example, see Chuang et al. J. Lab. Clin. Med. 92, 483-496 (1978). Thus, any solid support coated with passively adsorbed protein is at risk of protein desorption when the support is exposed to high levels of protein such as that found in blood or serum fractions. Trace amounts of certain proteins in blood irreversibly bind to "contact activation" surfaces and can even displace or cause removal of protein previously adsorbed onto these surfaces (Biocompatible Polymers, Metals, and Composites (1983) p. 82).
Although poorly understood, the interaction of chemical substances with solid surfaces is important to the performance of diagnostic tests. Most fluids that contact surfaces used in these assay kits contain proteins. These proteins bind to and cover up sites on the vessal walls and on the solid phase support materials used for separation. Supports having different compositions are susceptible to this phenomenon in different ways. Complement activating supports are materials that activate the blood complement cascade system of plasma. Many materials having this property have been studied. Examples are glass, diamataceous earth, kaolin, crystals, soluble polymers such as dextran sulfate, crude preparations of collagen, microparticles and micelles such as sulfatides. The common features of these materials are negative charge and a high molecular mass (Griep et al. Biochem. 25, 21, 6688-6694 1986). The blood complement cascade system of plasma is comprised of proteins that interact with complement activating surfaces and/or each other. One result of the contact of complement activating support with plasma protein is clot formation.
Most medical diagnostic procedures require contact of the solid phase with blood or serum fractions. The solid supports used in such procedures are thus unusually susceptible to protein adsorption/desorption phenomena discussed above. Because of this blood component induced desorption problem associated with use of coated glass surfaces prepared by passive adsorption, tedious covalent coupling techniques have heretofore been used to adsorb molecular binding partners to glass. Any method that both overcomes the need for covalent coupling and eliminates or substantially reduces adsorption/desorption interference is therefore an important enhancement to the art.
The present invention provides certain proteins having exceptional affinities for contact activating surfaces (surface active) that can be used to link other proteins or binding substances to supports having these surfaces such that they remain bound to such supports in the presence of blood or serum. These surface active proteins are immune to desorption from complement activating supports in the presence of blood or blood fractions because they substantially resemble blood protein that participates in the blood complement activation system and are not displaced by these same proteins. Descriptions of these proteins and details of using them are disclosed herein.